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Published online 25 April 2006
Published in Crop Sci 46:1245-1252 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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PLANT GENETIC RESOURCES

Reaction to Three Races of Fusarium Wilt in the Phaseolus vulgaris Core Collection

Mark A. Bricka,*, Patrick F. Byrnea, Howard F. Schwartzb, J. Barry Ogga, Kristin Ottob, Amy L. Falla and Jeremy Gilberta

a Dep. of Soil and Crop Sciences, Colorado State Univ., Fort Collins, CO 80523
b Dep. of Bioagric. Sciences and Pest Management, Colorado State Univ., Fort Collins, CO 80523

* Corresponding author (Mark.Brick{at}colostate.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Seven races of Fusarium oxysporum f. sp. phaseoli Kendrick and Snyder (Fop) cause Fusarium wilt (FW) disease of common bean (Phaseolus vulgaris L.). FW occurs worldwide and has recently become a serious disease in the central and western USA. The objectives of this research were to identify new sources of resistance to Fop, characterize the Central/South American (CA/SA) Phaseolus Core Collection for reaction to races 1, 4, and 5 Fop and determine if a previously reported sequence characterized amplified region (SCAR) molecular marker would be useful to identify resistance among accessions that make up the CA/SA Core Collection. Seedlings from the CA/SA Core Collection were screened for reaction by a root dip inoculation procedure. Among accessions evaluated for reaction to race 1 Fop, 21 were resistant, 47 intermediate, and 126 susceptible. Fifteen accessions were resistant to race 4, 61 intermediate, and 114 were susceptible. Nine accessions were resistant to both races 1 and 4, and five of the nine (PI 207373, PI 307802, PI 308908, PI 309877, PI 310842) were also resistant to race 5 Fop. All accessions resistant to races 1, 4, and 5 of Fop were characterized as Middle American, which suggests that resistance to multiple races of Fop should be more prevalent in germplasm from Middle America. The SCAR marker previously developed to identify a quantitative trait locus (QTL) associated with resistance to Fop was not associated with resistance in the Core Collection.

Abbreviations: ASI, average severity index • CA/SA, Central/South American • Fop, Fusarium oxysporum f. sp. phaseoli • FW, Fusarium wilt disease • PCR, polymerase chain reaction • PPD, photoperiod sensitive • RAPD, random amplified polymorphic DNA • SCAR, sequence characterized amplified region


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
FUSARIUM WILT of common bean, caused by Fusarium oxysporum f. sp. phaseoli Kendrick and Snyder (1942), has become an important disease in portions of the central High Plains and western USA. The disease causes rapid yellowing of foliage, followed by defoliation and ultimately plant death. FW was first identified on common bean in the USA in 1929 (Harter and Weimer, 1929) and in the central High Plains in 1988 (Schwartz et al., 1989). FW is distributed worldwide including Brazil, Colombia, Peru, Costa Rica, Italy, Spain, Greece, the Netherlands, and central Africa (Ribeiro and Hagedorn, 1979a; Armstrong and Armstrong, 1981; Puhalla, 1985; Elias et al., 1993; Diaz-Minguez et al., 1996; Alves-Santos et al., 1999; Buruchara and Camacho, 2000). Plant stress due to soil compaction, poor soil drainage, and extremes in soil moisture or temperature accentuate disease symptoms and can cause severe yield loss (Schwartz et al., 1996). Increased incidences of the disease in the central High Plains and lack of effective cultural control measures led us to seek new genetic sources of resistance to Fop.

Seven pathogenic races of Fop have been identified worldwide on the basis of host response on a set of differential cultivars–lines (Woo et al., 1996; Alves-Santos et al., 2002). Woo et al. (1996) characterized five races as: Race 1 from South Carolina, USA; Race 2 from Brazil; Race 3 from Colombia; Race 4 from Colorado, USA; and Race 5 from Greece. Alves-Santos et al. (2002) characterized Races 6 and 7 from Spain.

Genetic resistance to specific races of Fop is controlled by both single genes (Ribeiro and Hagedorn, 1979b; Salgado et al., 1995; Cross et al., 2000) and quantitatively (Cross et al., 2000; Fall et al., 2001). Cross et al. (2000) reported that a single dominant gene controlled resistance to race 4 Fop found in pinto cultivars Fisher (Fisher et al., 1995) and Sierra (Kelly et al., 1990). Fall et al. (2001) reported partial resistance to race 4 Fop due to a QTL accounted for 63.5% of the phenotypic variance for resistance in the recombinant inbred line population A55 x Belneb-RR1. Fall (2001) developed a SCAR marker linked in coupling to the QTL and tested it on a broad range of germplasm and cultivars representative of races Durango and Mesoamerica. The SCAR correctly predicted Fop reaction in seven of 10 lines of race Mesoamerica but was not effective in germplasm representative of race Durango. Because the SCAR was reasonably effective in predicting Fop reaction in the preliminary evaluation of Mesoamerica germplasm, we wanted to evaluate the effectiveness of the SCAR marker in the entire CA/SA Core Collection.

Although some commercial common bean cultivars possess resistance to single races of Fop, few possess resistance to multiple races (Velasquez-Valle and Schwartz, 1997; Ogg et al., 2000). Therefore, there is a need to identify bean sources that confer resistance to multiple races of the pathogen to broaden the genetic base of common beans. According to Singh (2001), the genetic base of common bean cultivars is narrow, and "knowledge, access, and use of diversity available in cultivated and wild relatives are essential for broadening the genetic base of cultivars to sustain improvement." Ideally, new sources of resistance can be found in a Core Collection of a species. The concept of a Core Collection for a crop species, first proposed by Frankel and Brown (1984), is used to represent the genetic diversity present in a large working collection because the core constitutes a representative subsample of the total collection. Information from the Core Collection can also be used to expand and guide a larger more intensive search for accessions with resistance in the entire active collection. Miklas et al. (1999) used this approach to successfully search for new sources of resistance to white mold in common bean caused by Sclerotinia sclerotiorum (Lib.) de Bary. Because it was not possible to screen the entire collection of over 14 000 accessions of P. vulgaris maintained at the Western Regional Plant Introduction Station at Pullman, WA, we chose to screen the CA/SA Core Collection for reaction to three races of Fop and adaptation to local environmental conditions at Fort Collins, CO. The CA/SA Core Collection consists of 202 accessions that represent germplasm from race Mesoamerica of the Middle American (MA) gene pool and all three races of the Andean gene pool.

The objectives of this research were to identify new sources of resistance and characterize the CA/SA Phaseolus Core Collection for reaction to Races 1, 4, and 5 Fop and determine if the SCAR marker developed by Fall (2001) was associated with resistance in germplasm represented in this collection.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Seed from the CA/SA Phaseolus vulgaris Core Collection which contains 202 accessions was obtained from the USDA/ARS Western Regional Plant Introduction Station, Pullman, WA (http://www.ars.usda.gov/main/site_main.htm?modecode=53-48-00-00; verified 23 February 2006). Seed was used to screen each accession for reaction to races 1, 4, and 5 Fop, adaptation to the local environment in the field, and to classify each accession for origin (gene pool). Races 1, 4, and 5 Fop correspond to races from South Carolina, Colorado, and Greece, respectively (Woo et al., 1996). These races of Fop were chosen because races 1 and 4 are found in the USA, and race 5 is known to overcome genetic resistance to races 1 and 4 (Woo et al., 1996). Seedlings from each accession were screened by the root dip inoculation method as modified by Salgado and Schwartz (1993).

Field Evaluations
One hundred seventy-nine accessions of the CA/SA Core Collection were evaluated for four traits in a field environment at Fort Collins, CO. Twenty-three of the accessions failed to produce healthy plants and thus were not included in the field results. Traits evaluated included gene pool classification based on floral and pod morphology (Singh et al., 1991), growth habit according to Singh (1982), reaction to endemic races of rust based on visual presence and size of rust pustules according to Stavely et al. (1983), and physiological maturity. Physiological maturity of each accession was classified 87 d after planting into one of the following categories: Adapted, if plants had pods with physiologically mature seed; Late, if plants had open flowers and small undeveloped pods; Very Late, if floral buds but no flowers or pods were present; and Photoperiod sensitive (PPD), if there were no flowers or floral buds present. Seed from each accession was planted 3 June 2001 in a field nursery at Fort Collins, CO. Each accession was planted in one 7-m-long row spaced 0.61 m between rows. The field plot was maintained by overhead irrigation and fertilized at the recommended rate to assure optimum plant growth. On 31 August, notes were taken from 10 plants in each row. Accessions that did not have 10 plants were not included in the final results.

Fusarium oxysporum Isolates and Inoculum
All Fop isolates used in this study were previously assigned race designation by Woo et al. (1996). Stock cultures of Fop were used to prepare stock inoculum for screening (Salgado and Schwartz, 1993). Isolates of the three races of Fop were maintained at –4°C in culture tubes containing autoclaved, finely-sieved, sandy soil mixed with 2% (volume) powdered oatmeal, and 15% (v/v) distilled water. Two to 4 mg of stock inoculum were plated onto Petri dishes containing potato-dextrose agar, pH 5.6, and stored at room temperature to induce production of conidia. The day of inoculation, conidia from the cultures were suspended in distilled water, and the suspension was vortexed for 30 s then filtered through cheesecloth into an Erlenmeyer flask before use. The inoculum concentration was adjusted to 106 conidia mL–1 with a hemacytometer, and poured into a 1000-mL beaker and continually stirred for use as a root-dip inoculum.

Inoculation Procedure
Pathogenicity screening for all accessions was performed by the root-dip technique described by Pastor-Corrales and Abawi (1987), later modified by Salgado and Schwartz (1993). The procedure uses 16- to 20-d-old seedlings grown in a potting mix (50:50; vermiculite:peat by volume). The seedlings were removed from pots and the root system gently washed to remove excess potting mix and placed in tap water for 5 to 10 min. The distal 1/3 of the root system was clipped with a scissors and the root system was placed in the root-dip inoculum solution for 5 min. After inoculation, plants were transplanted to the same fresh sterile potting mix used for germination. The plants were watered 15 to 20 min after transplanting and every other day for the first 7 d, then as needed to maintain plant vigor. The plants were grown in a greenhouse maintained at approximately 16/32°C night/day, respectively. Supplemental lighting provided 13 h of light d–1.

Disease Evaluation
Twenty-one days after inoculation, the plants were evaluated for reaction to Fop by the CIAT disease severity scale. The CIAT scale rates plants according to percentage of leaf tissue with disease symptoms, such as drying, wilting or chlorosis, as follows: 1 = no disease symptoms and completely healthy; 3 = 10% of the leaf surface area showing disease symptoms; 5 = 25% of the leaf surface showing disease symptoms, as well as some whole plant stunting; 7 = disease symptoms on 50% of leaves, and severely stunted; and 9 = plant death. A plant is considered resistant if it scored 1 to 3, intermediate if scored 4 to 6, and susceptible if scored 7 to 9 on the CIAT disease severity score. Eight to 10 seedlings from each accession were evaluated. The mean for each accession of all plants evaluated are reported as the average severity index (ASI). In all experiments, inoculated resistant and susceptible parental checks were used to evaluate pathogenicity of the test and to confirm disease classification of known resistant and susceptible lines. The check entries were cultivar UI 114 and the line Lef-2RB that consistently rated susceptible (ASI > 8) and resistant (ASI < 3), respectively. In addition, two uninoculated plants from each accession were grown to evaluate the pathogenicity of the inoculum with the inoculated plants and determine the phenotype of each accession in the absence of disease symptoms.

SCAR Marker Development and Evaluation
A SCAR marker previously developed by Fall (2001) which was linked in coupling with a QTL for resistance to race 4 Fop was tested to determine if accessions in the Core Collection that amplified the SCAR had higher resistance than accessions that did not amplify the SCAR. The SCAR was developed from a standard RAPD (random amplified polymorphic DNA) reaction using DNA from the resistant parent and RAPD primer U20 (Operon Technologies, Alameda, CA). Primers were designed on the basis of the original RAPD primer sequence (10 bases) plus an additional 11 to 13 bases of the band sequence. Forward and reverse primer sequences were 5'-ACAGCCCCCATTGTGAATTGTAT-3' and 5'-ACAGCCCCCACACTTATGGCA-3', respectively. Primers were synthesized by Integrated DNA Technologies (Coralville, IA). Polymerase chain reactions were performed in microtiter plates in a reaction volume of 20 µL in a PTC-100 MJ thermal cycler (MJ Research Inc., Waltham, MA) with a heated lid. Final concentrations in the reaction mixtures were 200 ng mL–1 DNA template, 2 mM MgCl2, 100 µM each dNTP, 0.4 µM each primer, and 10 units mL–1 Taq polymerase. Cycling parameters were 30 cycles of 94°C for 1 min, 70°C for 30 s, and 72°C for 1 min. The SCAR products were separated on 1.5% (w/v) agarose gels and stained with ethidium bromide. The SCAR marker was tested in the A55 X Belneb 1 RIL population by Fall (2001) and cosegregation occurred with the resistant QTL.

We used the SCAR primers to evaluate 185 of the accessions from the CA/SA Core Collection. For these evaluations, DNA was extracted as described in Skroch and Nienhuis (1995) and PCR reactions were performed as described above.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Field Evaluations
Accessions varied for four traits evaluated in the field at Fort Collins, CO (Table 1). On the basis of morphological characteristics (Singh et al., 1991), 69 accessions could not be precisely classified into gene pool because they were photoperiod sensitive and did not produce mature flowers or fruiting structures critical to determine correct classification or the field stand had fewer than 10 plants. All photoperiod sensitive accessions had either Type III or IV growth habit, which occurs in both Andean and Middle American germplasm (White and Laing, 1989). Among accessions that could be classified to gene pool, 43 were classified as Andean and 90 as Middle American origin. Growth habit varied from I to IV. Most Andean accessions had Type I or IV growth habit, while most accessions classified as Middle American had Type II or III growth habit (Table 1).


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  		Table 1. Accession–entry, country of origin, gene pool, growth habit, relative maturity classification, reaction to endemic races of rust, and average severity index for reaction to Races 1, 4, and 5 of Fusarium oxysporum f. sp. phaseoli in the Phaselous vulgaris Core Collection. Only accessions resistant to Races 1 or 4 were screened for reaction to Race 5. ND indicates no data. Empty cells for disease reaction indicate that information was incomplete or missing.

 
Reactions to the endemic strains of rust in the field, according to the rating system developed by Stavely et al. (1983), at Fort Collins also varied among accessions. Infection levels were low and varied from 1 to 20%. One hundred forty-nine of the accessions had an immune response (rated 1), while none showed the resistant reaction (rated 2) expressed as necrotic leaf flecks. Only three accessions rated 3, indicative of the small pustule resistant reaction. The remaining accessions were susceptible with ratings 4 or higher. Because the rust infection was not severe, some lines may have escaped natural inoculation.

Physiological maturity among the accessions varied from adapted to very late. Eighty-three of the accessions produced physiologically mature pods by 31 August, suggesting that they were adapted to the local environment. The remaining accessions were classified as late, very late, or photoperiod sensitive. These results illustrate the genetic diversity for plant maturity that exists in the CA/SA Phaseolus Core Collection.

Disease Screening
Accessions varied in their reaction to the three races of Fop based on the modified CIAT root dip inoculation test (Table 1). The entire collection was screened for reaction to races 1 and 4 Fop, and accessions found to be resistant (ASI ≤ 3) to either races 1 or 4 were screened for reaction to race 5. Some of the accessions were not evaluated because they had poor germination or seedling vigor in the greenhouse test. Among the 194 accessions that were evaluated for reaction to race 1; 21 were classified resistant, 47 intermediate (ASI > 3 ≤ 7) and 126 susceptible (ASI ≥ 7) (Table 1). Among the 190 accessions screened for reactions to race 4: 15 were classified resistant, 61 intermediate and 114 susceptible.Nine accessions were resistant to both races 1 and 4. Seven of these nine accessions were characterized for gene pool in the field nursery, and all were classified as Middle American. Accessions PI 307802 and PI 309881 were not classified, however they are likely from the MA gene pool because they were collected in El Salvador and Nicaragua, respectively. These results indicate that resistance to Fop is present in the CA/SA Core Collection, and that resistance to both races 1 and 4 was limited to accessions from the MA gene pool.

Five of the nine accessions that were resistant to races 1 and 4 were also resistant to race 5 and included PI 207373, PI 307802, PI 308908, PI 309877, PI 310842 (Table 1). Two of these accessions were from Costa Rica, and one each from Colombia, El Salvador, and Nicaragua. Because all accessions resistant to the three races were classified as from the MA gene pool, resistance to multiple races of Fop may only occur in germplasm from the MA gene pool, and the search for resistance to multiple races of Fop in the entire collection should initially focus on germplasm representative of the MA gene pool.

SCAR Evaluation
In general, the SCAR was not associated with resistance among accessions in the CA/SA Core Collection. Among the 185 accessions evaluated, 59 accessions amplified the SCAR, while 126 did not (Table 2). ASI for Fop1 among lines in the entire collection that amplified the SCAR was 6.9, compared with 7.1 for lines that lacked the SCAR (P > 0.10). For reaction to race 4, mean ASI values were 6.8 and 6.6 (P > 0.10) for lines with and without the SCAR, respectively. When the collection was separated by Central or South American origin, accessions that differed for amplification of the SCAR also did not differ for reaction to either race 1 or 4 Fop. When accessions were separated based on country of origin, values for accessions from Nicaragua and El Salvador that did not amplify the SCAR were lower than the accessions that amplified the SCAR. These results are in contrast to what was expected because Fall (2001) reported that the SCAR was linked in coupling to the resistant QTL, and it was expected that presence of the marker would be associated with lower ASI. These results indicate that the SCAR marker would not have utility to identify resistant accessions in the CA/SA Core Collection.


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  		Table 2. Groups of accession by origin from the Phaseolus vulgaris Core Collection, occurrence of a SCAR marker, number of accessions in each group, and mean ASI values for reaction to Races 1 and 4 of Fusarium oxysporum fsp phaseoli.

 

   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Resistance to single and multiple races of Fop were found in the CA/SA Phaseolus Core Collection. Resistance was found primarily in accessions classified as Middle American, and resistance to all three races was only found in accessions that were representative of the MA gene pool. The SCAR marker developed in a RIL population linked to a QTL for resistance to race 4 Fop would not have utility for screening the CA/SA Core Collection. The identification of Middle American germplasm collected in Colombia, Costa Rica, El Salvador, and Nicaragua that possessed resistance to three races of Fop should be useful to conduct a more intense search for resistant germplasm collected in these countries by using additional traits such as seed size and color, growth habit, and others to identify useful germplasm that can be used in a breeding program for specific market classes.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Contribution of the Colorado State Agric. Experiment Station, Fort Collins, CO. Partial funding provided by the USDA National Plant Germplasm System.

Received for publication June 6, 2005.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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